CHRONIC NERVE COMPRESSION ALTERS SCHWANN CELL MYELINARCHITECTURE IN A MURINE MODELRANJAN GUPTA, MD,1 NIMA NASSIRI, BS,2 ANTONY HAZEL, MD,1 MARY BATHEN, BS,1 and TAHSEEN MOZAFFAR, MD1

1Department of Orthopaedic Surgery, University of California at Irvine, 2226 Gillespie Neuroscience Research Facility,Irvine, California 92697, USA

2Department of Biomedical Engineering, University of California at Irvine, Irvine, California, USA

Accepted 19 August 2011

ABSTRACT: Introduction: Myelinating Schwann cells compart-mentalize their outermost layer to form actin-rich channelsknown as Cajal bands. Herein we investigate changes inSchwann cell architecture and cytoplasmic morphology in anovel mouse model of carpal tunnel syndrome. Methods:Chronic nerve compression (CNC) injury was created in wild-type and slow-Wallerian degeneration (WldS) mice. Over 12weeks, nerves were electrodiagnostically assessed, andSchwann cell morphology was thoroughly evaluated. Results: Adecline in nerve conduction velocity and increase in g-ratio isobserved without early axonal damage. Schwann cells displayshortened internodal lengths and severely disrupted Cajalbands. Quite surprisingly, the latter is reconstituted withoutimprovements to nerve conduction velocity. Conclusions:Chronic entrapment injuries like carpal tunnel syndrome are pri-marily mediated by the Schwann cell response, wheredecreases in internodal length and myelin thickness disrupt theefficiency of impulse propagation. Restitution of Cajal bands isnot sufficient for remyelination after CNC injury.

Muscle Nerve 45: 231–241, 2012

To facilitate efficient transmission of nerveimpulses, Schwann cells in the peripheral nervoussystem (PNS) produce insulating layers of cytoplasm,called myelin, which ensheath large-caliber axons.The outermost, or abaxonal, layer of myelin is polar-ized to create a network of abundant cytoplasmflanked by patches of minimal cytoplasm. Ramon yCajal first identified this unique microstructureunderlying the plasma membrane of myelinatingSchwann cells and ascribed to them a role as trophicsupporters of the myelin sheath.1 These longitudinaland transverse cytoplasmic trabeculae are known asCajal bands and have long been the subject of muchinterest; however, little has been discovered abouttheir function, their role in facilitating Schwann cellmaturation, and their response to nerve injury.

For proper action potential propagation tooccur, adequate myelin thickness and Schwann cellinternodal length (IL) must be maintained. Recentstudies using periaxin-null mice have suggestedthat the Cajal bands facilitate the microtubule-

based transport of proteins and organelles neces-sary for Schwann cell elongation and maturation.2

Aberrations from this architecture coincide withirregularities in transmembrane signaling, specifi-cally within the dystroglycan–dystrophin axis,which is necessary for myelin maintenance.

Studies have focused on hereditary models of de-myelination as a means of investigating the relation-ship between impulse propagation, myelin thickness,IL, and Cajal band integrity. However, little hasbeen done to investigate the role of these factors inacquired injury. Entrapment neuropathies, such ascarpal and cubital tunnel syndromes, have beeneffectively reproduced in rat models throughchronic nerve compression (CNC) injury.3 Charac-terized by limited cytokine activation and delayedmacrophage recruitment,4 CNC injury differs dra-matically from the rapidly activated network of cyto-kines and macrophages associated with Walleriandegeneration (WD).5 Deficiencies in motor functionafter CNC injury are thought to result from long-term demyelination and decreases in IL.3,6

The current rat model is limited by inapplicabil-ity to transgenic studies. We generated a novelmouse model of CNC injury and evaluated differen-ces in Schwann cell function and architecturebetween wild-type and slow-WD (WldS) strains. Ourgoal was to elucidate the role that demyelinationplays in the development of CNC injury and to char-acterize changes in Schwann cell architecture that in-hibit the efficient propagation of nerve impulses.

METHODS

Mouse Model of Carpal Tunnel Syndrome. Twostrains of mice, 6 weeks old, were used: (1) thewild-type (WT) C57BL/6 (Harlan Laboratories,UK), which display normal WD; and (2) themutant C57BL/6-WLD/OLA/NHSD (Harlan Lab-oratories, UK), which display a neuroprotectivephenotype and abnormally slow WD.

Chronic nerve entrapment was introducedthrough a novel surgical approach. Mice were anes-thetized by intraperitoneal injection of ketamine/xylazine, and a dorsal gluteal-splitting approach wasused to isolate and mobilize the sciatic nerve. Tominimize the inflammatory response, all tubing wasplaced in a Petri dish the night prior to surgery,soaked in 70% ethanol, and allowed to dry in a

Abbreviations: ANOVA, analysis of variance; CMAP, compound muscleaction potential; CNC, chronic nerve compression; DAPI, 40,60-diamidino-phenylindole dichloride; DRP2, dystrophin-related protein 2; FITC, floures-cein isothiocyanate; IL, internodal length; PBS, phosphate-buffered saline;PFA, paraformaldehyde; PNS, peripheral nervous system; TRITC, phalloi-din tetramethyrhodamine isothiocyanate; WD, wallerian degeneration;WldS, slow-wallerian degeneration mouse; WT, wild-type

Correspondence to: R. Gupta; e-mail: ranjang@uci.edu

VC 2011 Wiley Periodicals, Inc.Published online in Wiley Online Library (wileyonlinelibrary.com).DOI 10.1002/mus.22276

Key words: Cajal bands, chronic nerve compression injury, demyelination,DRP2, Schwann cells

Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012 231

vacuum hood. A 3-mm biologically inert silastictube (Cole-Palmer, Vernon Hills, Illinois) with aninner diameter of 0.51 mm was placed atraumati-cally around the ipsilateral nerve below the level ofthe sciatic notch (Fig. 1A). Preliminary measure-ments of sciatic nerves in both WT and slow-WDmice (n ¼ 6) demonstrated an average diameter of0.378 6 0.029 mm. Consequently, after the experi-mental nerve was returned to the host bed, thetube readily glided along the nerve. Based on thecharacteristics of the tube polymer and pre-applica-tion processing, the tube was easy to remove at alltime-points of specimen harvest, and no grosschanges to nerve structure were observed (Fig. 1B).The contralateral sciatic nerve was isolated usingthe same technique and mobilized without place-ment of tubing in order to serve as a control.Wounds were closed in layers, and tension-free skinclosure was performed on all mice.

As positive controls for demyelination, a sepa-rate cohort of mice received crush injury. As previ-ously described,7 the right sciatic nerve was care-fully exposed, mobilized, and crushed immediatelydistal to its emergence from the gluteus maximususing hemostatic forceps for 30 seconds. The leftsciatic nerve was mobilized and returned to its hostbed without inducing crush. Approval for animaluse and all experimental procedures was obtainedfrom the institutional animal care and use commit-tee of the University of California, Irvine.

Electrodiagnostic Evaluation. Electrodiagnosticstudies of nerve conduction velocity were per-formed (n ¼ 10) preoperatively and serially atweekly postoperative time-points. Recordings ofboth the ipsilateral experimental and contralaterallimbs were gathered in vivo under ketamine/xyla-zine anesthesia on an electromyography machine(Sierra LT; Cadwell Laboratories, Kennewick,Washington). Motor conduction in the sciatic-tibialnerves was assessed by stimulating at the sciatic

notch and knee using a monopolar needle elec-trode. The reference for the stimulating electrodewas placed in the ipsilateral lumbar paraspinalmuscle. The compound muscle action potential(CMAP) from the tibial-innervated ankle plantarextensor muscle (tibialis anterior) was recorded byplacing subdermal electroencephalographic elec-trodes in the muscle approximately 2 mm abovethe heel. The reference-recording electrode wasinserted into the dorsal aspect of the foot, and theCMAP amplitude and motor nerve conductionvelocity were measured.

Light Microscopy and Morphometric Analysis. Preo-peratively, and at 2 and 6 weeks after injury, thesciatic nerve in the area of compression was har-vested from WT and WldS mice (n ¼ 4). Nerve seg-ments were coded for blind analysis and fixed in4% glutaraldehyde in 0.1 M phosphate-buffered sa-line (PBS, pH 7.4) at 10�C. After fixation, speci-mens were postfixed in 1% osmium tetroxide in0.1 M PBS, dehydrated in serial ethanol washes,and treated with propylene oxide. Samples wereincubated in a 1:1 propylene oxide and Eponresin, and then transferred to Epon resin. Speci-mens were transferred to Beem flat-embeddingmolds and baked at 60�C for 24 hours. Blocks werecut with an ultramicrotome to obtain 1-lm sectionsand stained with toluidine blue. Whole nerve mapsof cross-sections were captured at 100� magnifica-tion using an inverted microscope (1X71; OlympusImaging America, Inc.). The g-ratios were calculatedas the ratio of axon diameter to the total fiber di-ameter for 1000 axons per group per time-point.Total axon counts, and number of myelinatedaxons were evaluated in uninjured and injured WTsamples for over 1000 axons per time-point. Distri-butions of axon diameter (d) were also evaluated inuninjured and compressed specimens, and fiberswere categorized as either small (d � 2 lm), me-dium (2 lm < d < 4 lm), or large (d � 4 lm). All

FIGURE 1. Mouse model of CNC injury. (A) A bioinert silastic tubing is placed around the sciatic nerve, providing chronic compression

and mechanical stimulation in a manner consistent with entrapment neuropathies. (B) A representative image of whole nerve cross-

section from a compressed specimen is shown. No gross changes in nerve structure could be seen at any of the time-points of speci-

men harvest.

232 Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012

measurements were taken using SlideBook software(Intelligent Imaging Innovations).

IL Measurements. Contralateral and ipsilateral sci-atic nerves were harvested at postoperative time-points (n ¼ 4). After fixation in glutaraldehyde,samples were postfixed in 1% osmium tetroxide at37�C for 2.5 hours. Each sample was then seriallytreated for 24 hours with 44%, 66%, and 100%glycerin at 37�C. Under a surgical microscope, sin-gle myelinated fibers were teased apart using ultra-fine forceps. Over 25 fibers were teased per nervesample for measurements of IL. For compressednerve samples, IL was measured in the zone ofinjury. IL was measured with Visiopharm Integra-tory System software (Visiopharm, Denmark).

Tissue Preparation for Immunohistochemistry. Atthe 2-, 4-, 6-, and 12-week postoperative time-points, mice (n ¼ 4) received intracardiac perfu-sion with 4% paraformaldehyde in 0.1 M PBS (pH7.4). Ipsilateral and contralateral sciatic nerveswere harvested, postfixed in 4% paraformaldehyde(PFA) for 30 minutes, and stored at �80�C. Undera surgical microscope, the endoneurium and peri-neurium were stripped, and myelinated fibers weremanually teased using ultrafine forceps. Previousstudies suggested that myelin abnormalities afterchronic injury occur initially on the outermostfibers.8 Thus, we selected these fibers for evalua-tion through immunohistochemistry.

Teased fibers were blocked and permeabilizedwith 0.1% Triton X-100 and 5% fish skin gelatin(Sigma) in PBS for 1 hour at room temperature.Primary antibodies were applied in the same block-ing/permeabilizing solution overnight at 4�C. Sub-sequently, fibers were washed in PBS with 0.1% Tri-ton X-100. Secondary antibodies were applied inblocking/permeabilizing solution for 3 hours atroom temperature. After several washes, excess PBSwas removed, and fibers were mounted in Vecta-Shield (Vector Laboratories). Images were acquiredusing an inverted microscope (Olympus 1X71).

Primary/Secondary Antibodies and Dyes. The follow-ing antibodies and dyes, sources, and dilution wereused: rabbit anti-DRP2 (a gift from P.J. Brophy, Uni-versity of Edinburgh, Edinburgh, UK; 1:200); fluo-rescein isothiocyanate (FITC) and rhodamine-conju-gated phalloidin (Sigma; 1:400); mouse anti-S100(Chemicon; 1:600); goat anti-rabbit FITC (JacksonImmunoResearch; 1:400); goat anti-rabbit tetrame-thylrhodamine isothiocyanate (Jackson ImmunoRe-search; 1:400); and 40,60-diamidino-2-phenylindoledihydrochloride (DAPI; Sigma; 4 lg/ml). Teasedsamples were immunostained to determine thestructural integrity of Cajal bands using mouse anti-S100, phalloidin–tetramethyrhodamine isothiocya-

nate (TRITC), and DRP2. As previous studies haveutilized F-actin to outline the location of Cajalbands, double-immunostaining using phalloidin-FITC and DRP2 was completed to visualize Cajalbands and the appositions they border.

Morphological Analysis and F-Ratio. Using ImageJ(NIH), DRP2 and phalloidin staining wereadjusted (after red–green–blue color split) usingthe threshold function. The threshold (in blackand white) was set arbitrarily for each image tomatch most closely the size and shape of trabecu-lae and patches. The Pearson R coefficient was cal-culated (n ¼ 20, from 4 animals) at each time-point using the Intensity Correlation Analysis plu-gin. The combination of channel color was estab-lished as TRITC vs. FITC, and pixels were analyzedin both channels for overlap. A perfect correlationoccurs at R ¼ 1, and values approaching 1 indicatereliable colocalization.

Schwann cell compartmentalization at the light-microscope level was determined as previouslydescribed.9 Calibrated images of the completeSchwann cell volume immunostained with antibod-ies against DRP2 and phalloidin-FITC wereobtained. At least 20 fibers from 4 animals wereanalyzed. The F-ratio, defined as the ratio of areaoccupied by cytoplasm-rich Cajal bands (F-actin sig-nal) to DRP2-filled plaques, was calculated inchronically compressed nerve segments. DRP2staining was adjusted using the threshold function.DRP2 patches have defined edges, and the use ofa different threshold for each image does not addsignificant error but was necessary because of dif-ferences in overall DRP2 staining intensitiesbetween samples processed at different time-points. The area occupied by the DRP2 signal wasmeasured using the Analyze Particles option. TheCajal bands/trabeculae area was defined as regionof the Schwann cell compartment lacking DRP2staining. These open cytoplasmic regions were esti-mated by measuring the whole Schwann cell areaand subtracting the corresponding DRP2 area.

Statistical Analysis. An equal number of samplesand data points were obtained from experimentaland control groups for each time-point. Electrophysi-ological measurements and g-ratio data areexpressed as mean 6 SEM and were evaluated usingthe Student t-test and one-way analysis of variance(ANOVA) followed by Tukey–Kramer post hoc test.Differences were considered significant at P < 0.01.

RESULTS

CNC Injury Causes Sustained Decreases in Nerve Con-

duction Velocity. For an animal model of compres-sion neuropathy to re-create the human condition,there must be a progressive decline in nerve

Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012 233

conduction velocity in the area of compression. Todetermine the degree of neuropathy resultingfrom CNC injury, we conducted serial electrodiag-nostic evaluations throughout a 12-week timecourse (Fig. 2). In WT mice, conduction velocitydecreased progressively after CNC injury from abaseline of 51.5 6 1.6 m/s to 37.5 6 2.5 m/s at 6weeks after injury. After the 6-week time-point, theconduction velocity plateaued and remained con-sistently low through the 8-, 10-, and 12-week time-points. To confirm that this decline resulted pri-marily from demyelination rather than axonaldamage, we analyzed CMAP amplitudes at eachtime-point. CMAP amplitudes represent all theaxon bundles comprising the nerve. A decrease inthe total number of axons resulting from nervedamage would cause a reduction in the evoked am-plitude. There were no statistically significant dis-crepancies in amplitude between experimental andcontrol groups at any of the time-points observed.

To further assess the role of axonal damage inthe progression of CNC injury, we evaluated nerveconduction velocities in mice with delayed-onsetWD. The WldS mouse is a spontaneously occurringmutant with a triplication of the fusion geneUbe4b/Nmnat and a phenotype of axon protectionin both the central and peripheral nervous sys-

tems.10,11 If CNC injury induces early axonalpathology, such a finding would not be evident inthe mutant strain until later time-points. AfterCNC injury, WldS mice exhibited an immediateand progressive decline in conduction velocity,similar to their WT counterparts. Non-compressed(contralateral) nerves maintained a baseline con-duction velocity of 56.1 6 3.61 m/s. As early as 1week after CNC injury, average velocity declinedand reached a plateau of 34.6 6 6.38 m/s by the4-week time-point (Fig. 2C). There were no signifi-cant discrepancies in CMAP amplitude betweenthe compressed and non-compressed groups.

CNC Injury Induces Changes in Fiber Size and Myelina-

tion. To morphometrically evaluate axonal andaxoglial integrity after CNC injury, we comparedtotal axon counts with the number of myelinatedaxons in uninjured and compressed nerve speci-mens from WT mice. No significant change inoverall axon numbers was observed between nor-mal samples and those harvested at the 2- and6-week time-points after CNC injury (Fig. 3A).Comparison of total axon counts versus the num-ber of myelinated fibers in each group demon-strated a statistically significant decline in myelin-ated axons at 2 and 6 weeks after CNC injury, with

FIGURE 2. Electrodiagnostic assessment of nerve conduction velocity and CMAP amplitude after CNC injury. (A) WT mice demon-

strate a progressive decline in nerve conduction velocity after chronic nerve compression injury of the affected nerve. (B) No significant

discrepancies in CMAP amplitudes could be seen in CNC and control groups. (C) Nerve conduction velocity in WldS mice after CNC

injury mirrors the trend observed in WT mice, demonstrating a progressive, sustained decline in the speed of impulse propagation

from 1 to 6 weeks after compression. (D) CMAP amplitudes remain unaffected in WldS mice.

234 Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012

more pronounced demyelination observed at thelater time-point (P < 0.01).

We next sought to evaluate changes in axonfiber diameter at various time-points after CNCinjury. The diameters of 1000 axons per time-pointwere measured and categorized as small (d �2 lm), medium (2 lm < d < 4 lm), or large (d �4 lm) (Fig. 3B). A significant increase wasobserved in the number of small-sized fibers by6 weeks after CNC injury, which coincided withdecreases in the proportion of large-sized fibers atthe same time-point (P < 0.001). Although thefraction of medium-sized axons fluctuated betweennormal and 2- and 6-week post–CNC injury sam-ples, these changes were not statistically significant.

CNC Injury Induces Sustained Decreases in Myelin

Thickness. To determine the effect of CNCinjury on myelin thickness, we calculated the g-ratio in large-caliber fibers from WT and WldS

nerve samples (Fig. 4G). Average g-ratio valuesfor WT uninjured nerves approximated 0.62 60.0012. We found a statistically significant eleva-tion in this value 2 weeks after compression (P <0.001). Six weeks after CNC injury, g-ratio valuespeaked (0.792 6 0.0076) (Fig. 4A–C, H). Suchelevation in the g-ratio corresponds to progres-sive myelin thinning. In WldS mice, the average g-ratio on the control side resembled the WTcounterpart, with a value of 0.62 6 0.0008. Aver-age values increased progressively after CNCinjury, peaking at 0.76 6 0.0008 by the 6-weektime-point (Fig. 4D–F, H).

As positive control, we measured changes inmyelin thickness after acute crush injury. In theWT mouse, sciatic nerve crush caused a sharpincrease in the average g-ratio that peaked 2 weeksafter injury and approached baseline values 6weeks after injury. Due to the neuroprotective phe-

notype of WldS mice, the average g-ratio remainednormal 2 weeks after nerve crush, and it elevatedin a delayed fashion 6 weeks after injury (Fig. 4H).

Decrease in IL Over Time After Chronic Compression

Injury. In conjunction with myelin thickness, ILalso affects the speed of impulse propagationalong the axon. Previous studies have demon-strated a correlation between decreased nerve con-duction velocity and IL,9,12 corroborated byincreases in nodal frequency in various models ofperipheral neuropathy.13 We sought to determinewhether CNC injury affects the length to whichSchwann cells can elongate. Analysis of singleteased nerve fibers from sciatic nerves of WT miceshowed a significant decrease (P < 0.0001) in ILover a 12-week time course (Fig. 5). Baseline ILsfor teased fibers approximated 633.5 6 15.4 lm.Two weeks after compression, ILs decreased to74.8% of normal, declining further to 56.6% ofnormal 6 weeks after CNC injury. IL remainedshortened 12 weeks after injury. After CNC injury,Schwann cells were unable to properly elongateand form internodes of normal length.

Actin Cytoskeleton in Outermost Cytoplasmic Layer Is

Interrupted After CNC Injury. Fluorescently labeledphalloidin toxin binds to and labels filamentousactin in the cell cytoskeleton.14 As Cajal bands arelargely comprised of a network of filamentousactin, we assessed morphological changes in micro-structure along the length of teased nerve fibers bystaining with phalloidin-FITC (Fig. 6, left). Immu-nohistochemistry revealed a dramatic disturbanceto Cajal bands immediately after CNC injury. Spe-cifically, the regular pattern of actin channels wasseverely disrupted 2 weeks after injury. Quite sur-prisingly, partial reconstitution of this actin scaf-fold became evident at the 6-week time-point;although irregular in pattern, a discrete network of

FIGURE 3. Morphometric evaluation of overall axon number, myelination, and fiber-size distribution. (A) Overall axon counts remain

consistent between the normal, 2-week, and 6-week CNC injury groups. Significant decreases in number of myelinated axons are

observed at both the 2- and 6-week time-points (P < 0.01). (B) Evaluation of fiber-size distribution reveals a significant increase in the

proportion of small-diameter fibers and a corresponding decrease in large-diameter fibers after week 6 after CNC injury (P < 0.001).

Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012 235

Cajal bands was identifiable. Twelve weeks afterinjury, the integrity of the actin scaffold resembleduninjured specimens: Cajal bands outlined apposi-tions of similar shape and size, and were symmetri-cal in pattern. Immunostaining of teased fibers forthe Schwann cell cytoplasmic protein S100 (Fig. 6,right) confirmed the pattern of Cajal band disrup-tion and subsequent reconstitution after CNCinjury.

Cajal Band Disorganization Compromises Apposition

Integrity. Currently, only one intracellular marker,DRP2, has been identified as being uniquely local-ized to the cytoplasmic appositions that are out-lined by Cajal bands.2 Using this marker, wesought to evaluate the spatiotemporal interplaybetween Cajal bands and the localization of DRP2to cytoplasmic appositions. Immunostaining forDRP2 in uninjured samples revealed deposits ofuniform shape and size and of a regularly repeat-

ing pattern throughout the Schwann cell inter-node (Fig. 7). Two weeks after CNC injury, DRP2clusters were disrupted, and diffused staining wasobserved throughout the length of the internode.Similar to the pattern of disruption and reconstitu-tion observed in Cajal bands, a gradual reconver-gence of DRP2 into discrete plaques occurred atlater time-points. Six weeks after injury, DRP2localized to form appositions, although the shapeand size of plaques was irregular and incomplete.By 12 weeks post–CNC injury, DRP2 stainingapproximated that of uninjured samples, with pla-ques of regular pattern and shape.

Double immunofluorescence confirmed thatthe pattern of DRP2 delocalization and conver-gence to cytoplasmic appositions coincided tempo-rally with the disruption and subsequent reconsti-tution of Cajal bands (Fig. 8). To assess the degreeof overlap between DRP2 and phalloidin-FITC, wedetermined colocalization levels using the Pearson

FIGURE 4. Myelin thinning post–CNC injury in wild-type (top row) and WldS (bottom row) mice. (A, D) Uninjured samples demonstrate

normal myelin thickness. (B, E) Two weeks after CNC injury, both WT and WldS mice exhibit decreases in myelin thickness (arrows).

(C, F) Demyelination is sustained 6 weeks after injury in both mouse models (scale bar ¼ 10 lm). (G) Myelin thickness was quantita-

tively assessed by calculating g-ratios. (H) Uninjured samples exhibit baseline g-ratios of 0.623 6 0.0012 (WT) and 0.62 6 0.0008

(WldS). CNC injury induces significant increases in g-ratio (P < 0.001). In WT mice, crush injury (control) induces an immediate spike

in g-ratio, coinciding with the rapid onset of Wallerian degeneration, which returns to baseline values by 6 weeks. WldS mice demon-

strate the neuroprotective phenotype through a delayed spike in g-ratio after crush injury.

236 Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012

R coefficient. As expected, uninjured samplesdemonstrated minimal overlap between Cajalbands and appositions. Postinjury, this overlapspiked most dramatically at the 2-week time-pointand decreased progressively thereafter, and thedegree of colocalization approximated near-nor-mal values 12 weeks after injury (P < 0.01) (Fig.8B). This finding is unique from investigationsinto genetic models of demyelinating neuropa-thies and may be attributable to the dual proc-esses of demyelination and remyelination occur-ring concurrently.

To quantitate the changes in cytoplasmic mor-phology that were observed after CNC injury, wecalculated the F-ratio, defined as the ratio of theinternodal area occupied by cytoplasmic-rich Cajalbands to the internodal area occupied by DRP2-positive appositions, in normal and chronicallycompressed nerve segments. Normal nerves exhib-ited an average F-ratio value of 1.39 6 0.25,indicating an approximately equal distributionbetween the areas occupied by Cajal bands andappositions. The F-ratio spiked to a maximum of4.46 6 0.55 at 2 weeks after injury (P < 0.01).

FIGURE 5. Schwann cell elongation is hindered after CNC injury. ILs were measured for mice (n ¼ 4) at 2, 6, and 12 weeks post–CNC

injury. (A) Representative images of uninjured teased nerve fiber (top) compared with 6-week compressed fiber (bottom) (scale bar ¼100 lm). (B) Quantification of IL demonstrates a progressive decrease in IL that is sustained to the 12-week time-point (P < 0.0001).

FIGURE 6. Cajal bands dissociate after CNC injury, but are reconstituted at later time-points. In the left column, uninjured nerves dis-

play an intact network of Cajal bands as identified by immunostaining with phalloidin-FITC (green). Cajal bands outline appositions of

similar shape and size (arrowheads). At 2 weeks after CNC injury, Cajal bands are severely disrupted, and appositions cannot be

determined. Six weeks after injury, an irregular pattern of distinct bands is visible, outlining appositions that are irregular in shape and

size. By 12 weeks, a more regular pattern of Cajal bands is observed, more closely approximating uninjured samples. Immunostaining

with phalloidin also reveals Schmidt–Lanterman incisures (asterisks). In the right column, immunostaining of teased fibers for the

Schwann cell cytoplasmic protein S100 (red) confirms the pattern of Cajal band disruption and subsequent reconstitution after CNC

injury (scale bar ¼ 100 lm).

Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012 237

Subsequent time-points revealed a return to near-baseline values, with average F-ratios for the 6- and12-week time-points equaling 2.36 6 0.65 and 1.866 0.21, respectively (P < 0.01) (Fig. 8C).

DISCUSSION

The goals of this study were threefold. As the pre-viously described rat model of CNC injury repre-sents a reliable yet scientifically limited injurymodel for the study of entrapment neuropathies,we first sought to develop a mouse model of CNCinjury. Second, we sought to evaluate the role ofWallerian degeneration in this injury model. Ourthird aim was to assess morphological changesresulting from CNC injury, specifically with respectto myelin thickness, IL, and the integrity of theCajal band network.

Prior investigations into chronic compressioninjuries have commonly utilized rat animal mod-els.15–19 However, such models are limited fromthe use of transgenic and knockout techniques.We thus sought to establish an easily reproduciblemouse model wherein CNC injury could be moreaggressively investigated. The shared hallmark ofall entrapment neuropathies is a progressive andsustained decline in nerve conduction velocity

FIGURE 7. DRP2 complex is disrupted after CNC injury but is

reconstituted at later time-points. Uninjured nerves immuno-

stained with DRP2 (red) reveal distinct, symmetric plaques

along the internode. DRP2 deposits are disrupted 2 weeks after

CNC injury. By 6 weeks, DRP2 appears to converge to form

plaques, although the shape, size, and organization of these

plaques are irregular and often incomplete. Twelve weeks after

injury, the samples display more uniformly filled DRP2 plaques,

although their shapes remain non-uniform and oblong (scale

bar ¼ 100 lm).

FIGURE 8. Immunohistochemistry reveals a correlation between the integrity of Cajal bands and DRP2 localization to cytoplasmic

appositions. (A) Uninjured nerve fibers immunostained with phalloidin-FITC (green) and DRP2 (red) display an organized pattern of

DRP2 localization and regular Cajal band pattern, which becomes disrupted 2 weeks after injury and is gradually reconstituted at the

6- and 12-week time-points. (B) To assess the degree of colocalization, the Pearson R coefficient was calculated at each time-point (n

¼ 20, from 4 animals). Overlap between DRP2 and phalloidin–FITC is lowest among uninjured nerve samples. Dissociation of Cajal

band structure and the DRP2 complex at 2 weeks post–CNC injury corresponds to the greatest degree of overlap. This overlap

decreases at 6 and 12 weeks after CNC injury, coinciding with the reconvergence of DRP2 to discrete appositions and the constitution

of Cajal bands. (C) F-ratio measurements confirm gross disruptions to myelin morphology after CNC injury followed by gradual but

incomplete restitution to baseline values (scale bar ¼100 lm). F-ratios are abnormally high 2 weeks after injury and gradually return

to near-baseline values 6 and 12 weeks after injury (P < 0.01).

238 Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012

postinjury. Our electrodiagnostic data demonstratethis trend, as decreases in nerve conduction veloc-ity were sustained throughout the 12-week timecourse. Analysis of CMAP amplitudes demonstratethat demyelination, rather than axonal damage,plays the primary role in diminishing nerve con-duction velocity. Our mouse model thus exhibitsthe classical hallmarks of entrapment neuropathy.As our electrophysiological findings suggesteddemyelination in the absence of axonopathy, wesought to characterize this phenomenon morpho-metrically through counts of total axons and my-elinated axons. As expected, there were no signifi-cant changes in total axon numbers; however,demyelination was observed at both the 2- and6-week time-points. This finding supports ourhypothesis that the Schwann cell response afterCNC injury plays the primary role in the develop-ment of the ensuing neuropathy.

Although overall axon numbers did not changebetween uninjured and experimental samples, weobserved a decrease in the proportion of large-diameter fibers at 6 weeks post–CNC injury thattemporally correlated with an increase in the pro-portion of small-diameter fibers. Previous studiesin rat models of entrapment neuropathy have illus-trated that, after CNC injury, a phenotypic switchoccurs in neurons within the dorsal root gangliathat is characterized by increased sprouting, ele-vated expression of the small-fiber markers calcito-nin-gene–related peptide and isolectin B4, andcoinciding decreases in the large-fiber marker neu-rofilament 200.20 Consequently, the increases insmall-diameter axons and decreases in large-sizedfibers we observed may be a function of theincreased sprouting that occurs after CNC injury.

We next assessed whether, in conjuction withdemyelination, the process of Wallerian degenera-tion plays a significant role in the development ofCNC injury. Naturally occurring mutant WldS miceexpress a fusion protein known to delay WD afterneuronal injury and demonstrate a multifacetedneuroprotective phenotype.21 We hypothesizedthat, if WD did play a role in mediating the neuro-pathology, the decline in nerve conduction velocitywould be delayed in WldS mice. Electrophysiologi-cal evaluation of WldS mice mirrored that of theirWT counterpart and demonstrated an immediatebut progressive decline in nerve conduction veloc-ity that was sustained throughout all time-points.No significant discrepancies in CMAP amplitudeswere observed between injured and noninjuredgroups. These findings strongly suggest that axonaldamage and WD are not key players in the patho-genesis of CNC injury, and rather substantiateSchwann cells as the primary agents of the ensuingneuropathy.

We next sought to examine the morphologicalchanges that occur after CNC injury in myelinatingSchwann cells. The g-ratio calculations confirmeda significant progressive thinning of the myelinsheath after injury in both WT and WldS mice. Inthe absence of WD, the same pathological stateensues. Increases in g-ratio occur over a similartime course and exhibit a similar progressive trendas the observed decline in nerve conduction veloc-ity. Sciatic nerve crush was used as a positive con-trol to which the trends in g-ratio after CNC injurywere compared. After crush, the average g-ratiovalue increased sharply and re-approximated base-line values by the 6-week time-point, indicatingeffective axonal regeneration and remyelinationafter the initial insult. This differed dramaticallyfrom the progressive rise in g-ratio observed afterCNC injury, which remained elevated at the 6-weektime-point. Such findings confirm the existence ofintrinsic differences between the pathogenesis ofCNC injury and acute nerve injury. Specifically, thesecondary role of axonal trauma in the CNC injurymodel makes it a primarily Schwann cell–mediatedinjury state.

In conjunction with myelin thickness, Schwanncell IL is a major determinant of the efficiencywith which action potentials are propagated alongthe axon. We found dramatic decreases in IL 2weeks after CNC injury in both WT and WldS mice.Similar to observations on myelin thickness, thedecline in IL occurred progressively and plateauedat later time-points. Shortening of the internodecoincided temporally with changes in g-ratio andnerve conduction velocity. Consequently, wepropose that decreases in myelin thickness andIL mediate the ensuing aberrations in impulsepropagation.

To further investigate changes in myelin archi-tecture, we evaluated the integrity of Cajal bandsafter CNC injury. Cajal bands are believed to pro-vide trophic support to the myelinating Schwanncell by facilitating the transport of critical proteinsand nutrients within the myelin sheath.22 Theyare thought to play an essential role in Schwanncell elongation and growth.12 A rigorous 12-weekimmunostaining work-up revealed a dramatic dis-ruption of Cajal bands as early as 2 weeks afterinjury, which coincided with dispersal of DRP2throughout the length of the internode. The F-ra-tio, defined as the ratio between the area occu-pied by Cajal bands and DRP2-filled appositions,increased dramatically, corresponding to disrup-tion of internodal architecture. These early find-ings support the theory that Cajal bands providetrophic support and that, in their absence,Schwann cells cannot elongate to appropriatelengths.

Schwann Cells Mediate Pathogenesis of CNC Injury MUSCLE & NERVE February 2012 239

Because Schwann cell internodes remain short-ened throughout the 12-week time course, we hadinitially expected Cajal bands to remain disrupted.Quite surprisingly, our results for the 6- and 12-week time-points revealed a progressive reconstitu-tion of Cajal bands. F-ratio values reflected thesefindings and indicated a gradual but incompleteregression to baseline levels of localization. A plau-sible explanation for this phenomenon is that, in achronic injury model, such as CNC, mechanicalstimuli are consistently applied. Consequently, theopposing processes of demyelination and remyeli-nation occur simultaneously. Ultimately, the con-tinued presence of the mechanical stimuli mayresult in equilibrium between the opposing proc-esses of demyelination and remyelination. Thisalso may explain the observed plateau of nerveconduction velocity, g-ratio, and ILs. Alternatively,the restitution of Cajal bands, despite the preva-lence of diminished IL, may indicate that otherfactors play a role in perpetuating the neuropatho-logical state. Chronic ischemia may play a factor aswell, as hypoxia and limited nutrient delivery arebelieved to play a role in entrapment injuries.23

CNC injury mimics the pathogenesis and clini-cal manifestations of entrapment neuropathies,such as carpal and cubital tunnel syndromes. Stud-ies have suggested that the neuropathology thatfollows CNC injury is induced by changes in theinteraction between myelinating Schwann cells andtheir extracellular environment.4,20,23,24 Mechani-cal stimulation via shear stress is known to alterthe basal lamina and extracellular matrix, affectingmajor signaling proteins such as fibronectin andthe family of laminins.25–27 Cell surface receptorsfor these extracellular components, such as integ-rins and the dystroglycan complex, consequentlyprovide Schwann cells with mechanosensitive prop-erties.28,29 Given these findings, it is probable thatchanges incurred in the extracellular microenvir-onment as a result of CNC injury are internalizedby Schwann cells. Studies have demonstrated astriking number of shared signaling molecules,such as the a6b4 and a6b1 integrins and DG,30,31

and overall pathways, such as extracellular signal–related kinase 1 and 2,32–34 between CNC injuryand other demyelinating neuropathies, includingCharcot–Marie–Tooth disease, multiple sclerosis,and leprosy.34–36 Our current ongoing investiga-tions are aimed at elucidating the changes to theextracelluar microenvironment after CNC injury,with a greater goal of uncovering the moleculardeterminants that cause the altered myelin archi-tecture observed in this study.

The authors thank Dr. Peter J. Brophy (Center for Neuroscience,University of Edinburgh) for the generous donation of their DRP2

antibody. This project was funded by a grant from the NationalInstitutes of Health (NIHNINDS 2R01NS049203).

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